Longshan lily bulbil polysaccharide and its preparation method and application

Abstract

The present invention discloses a Longshan lily bulbil polysaccharide, its preparation method, and application, belonging to the field of plant extraction technology. The polysaccharide has a total sugar content greater than 94%, a monosaccharide composition of mannose: rhamnose: glucose: galactose: galacturonic acid = 5.12-5.48: 7.04-7.79: 61.55-62.38: 17.37-18.14: 7.28-7.91, and a weight-average molecular weight of 183,916-184,735 Da. The Longshan lily bulbil is treated with boiling water to inactivate the enzymes, decolorize and defatted, and the alcohol-insoluble matter is extracted with water. Starch is then removed using a thermostable amylase. The resulting filter residue is then microwave-digested with a sodium hydroxide solution, and the extract is dialyzed and extracted to produce the immunologically active Longshan lily bulbil polysaccharide. The polysaccharide can be used in food, functional foods, or pharmaceuticals, providing a promising approach for high-value applications of Longshan lily.

Description

Technical Field

[0001] The invention relates to the technical field of plant extraction, in particular to a Longshan lily bulbil polysaccharide and a preparation method and application thereof. Background Art

[0002] Longshan lily, a species of Lilium lilium in the genus Lilium of the family Liliaceae, is a protected geographical indication product in Longshan County, Hunan Province. With its jade-white color, slightly bitter flavor, and high nutritional value, Longshan lily has earned a high reputation both domestically and internationally. During its growth, Longshan lily produces numerous bulbils. To reduce nutrient consumption, these bulbils are typically removed before harvest. These bulbils are often discarded as by-products, resulting in a significant waste of resources. Longshan lily bulbils contain a significant amount of polysaccharides (over 16%). Effectively developing and utilizing these polysaccharides would significantly promote the comprehensive utilization of Longshan lily by-products, increase the added value of processed lily by-products, and promote the high-quality development of the Longshan lily industry.

[0003] In the prior art, the extraction of lily polysaccharides mostly uses lily petals, bulbs, etc. as raw materials, and adopts alcohol extraction and water extraction to extract water-soluble polysaccharides. For example, Chinese patent CN100572397A reports a method for preparing lily polysaccharides that enhance immunity, and extracts water-soluble polysaccharides with immunity from Lanzhou lily petals. Chinese patent CN114835831A discloses a lily polysaccharide extraction process from Changbai Mountain lily bulbs, which uses ultrasonic treatment + mechanical wall breaking to obtain a high extraction rate and better maintain the antioxidant activity of the polysaccharide. Chinese patent CN112812197A discloses a Lanzhou lily polysaccharide and its preparation method and application. The lily polysaccharide obtained by extracting, separating and purifying Lanzhou lily petals has significant anti-breast cancer and liver cancer activity. Chinese patent CN118530380A discloses a polysaccharide from lily bulbs of Longya lily and its preparation method and application. The lily polysaccharide obtained by ion exchange column purification has the effect of inhibiting the proliferation of harmful bacteria and promoting the proliferation of probiotics. There are few reports on the use of Longshan lily as a raw material for lily polysaccharides. Yang Zhaoxia et al. [Study on Optimization of Ultrasonic Extraction Process of Lily Polysaccharides by Response Surface Methodology, Guangzhou Chemical Industry, 2013, 41 (18): 52-55] used ultrasonic technology to extract Longshan lily polysaccharides and optimized the extraction process by response surface methodology.

[0004] Currently, there is no report on the extraction of polysaccharides from the bulbils of Lilium longshanense. Therefore, a method for preparing polysaccharides from the bulbils of Lilium longshanense is needed. Summary of the Invention

[0005] The purpose of the present invention is to provide a Longshan lily bulbil polysaccharide, a preparation method and an application thereof. Longshan lily bulbil is used as a raw material, and an alkali-soluble polysaccharide is obtained by boiling water withering, alcohol extraction and alkali dissolution. The polysaccharide has higher immune activity than water-soluble polysaccharides.

[0006] The invention provides a Longshan lily bulbil polysaccharide, the total sugar content of which is greater than 94%, the monosaccharide composition is: mannose: rhamnose: glucose: galactose: galacturonic acid = 5.12-5.48: 7.04-7.79: 61.55-62.38: 17.37-18.14: 7.28-7.91, and the weight-average molecular weight is 183916-184735 Da.

[0007] Preferably, the polysaccharide is extracted from fresh Longshan lily bulbils.

[0008] The present invention also provides a method for preparing the above-mentioned Longshan lily bulbil polysaccharide, comprising the following steps:

[0009] S1. Take Lilium bulbils, sterilize them with boiling water to inactivate enzymes, dry them, grind them, and pass them through a 300-500 mesh sieve to obtain Lilium bulbils powder;

[0010] S2, adding 5 to 10 times the amount of ethanol solution to the Longshan lily bulbil powder, decolorizing and defatting, filtering, and drying the filter residue to obtain Longshan lily bulbil alcohol-insoluble matter;

[0011] S3, adding water and thermostable amylase to the bulbilol-insoluble matter of Longshan lily, extracting at 75-85° C. for 100-200 minutes, filtering to obtain a residue and a filtrate, and setting the residue aside;

[0012] S4. Add sodium hydroxide solution to the filter residue obtained in step S3, perform microwave digestion, concentrate the filtrate by rotary evaporation, and dialyze the filtrate for later use;

[0013] S5. After the dialyzate is concentrated, it is extracted with a mixture of chloroform and n-butanol, the upper aqueous solution is collected, anhydrous ethanol is added, and the mixture is allowed to stand, followed by centrifugation to obtain a precipitate, which is then dried to obtain Longshan lily bulbil polysaccharide.

[0014] Preferably, in step S1, the time for sterilizing the enzyme is 90 to 120 seconds; preferably, the pulverization is performed in a low-temperature ultrafine pulverizer.

[0015] Preferably, the ethanol solution is an ethanol aqueous solution with a mass fraction of 95%.

[0016] Preferably, the decolorization and degreasing specifically comprises: treating at 40-50° C. for 2-6 hours, and repeating 3-6 times.

[0017] Preferably, in step S3, the volume of water added is 10-20 times the mass of the bulbilol-insoluble matter of the Longshan lily, and the amount of the thermostable amylase used is 0.2%-1% of the mass of the bulbilol-insoluble matter of the Longshan lily.

[0018] Preferably, in step S4, the volume of the sodium hydroxide solution added is 15-30 times the mass of the filter residue, wherein the concentration of the sodium hydroxide solution is 3wt%~6wt%.

[0019] Preferably, in step S4, the microwave digestion is specifically: extracting 2 to 4 times at a power of 400 to 800 W, each time for 20 to 40 minutes, cooling to room temperature, and filtering.

[0020] Preferably, in step S5, the volume ratio of chloroform to n-butanol in the chloroform and n-butanol mixture is 1:3-5.

[0021] Preferably, anhydrous ethanol is added to the solution so that the volume proportion of anhydrous ethanol is 75% to 80%.

[0022] Preferably, the standing temperature is 4° C. and the standing time is 8 to 12 hours.

[0023] Preferably, the drying temperature is 40-50°C.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention uses Longshan lily bulbils as raw materials, and first uses boiling water to briefly blanch the bulbils to inactivate enzymes such as oxidase in the bulbils, prevent enzymatic browning and other reactions that occur during the drying process to produce substances such as melanin, which requires prolonged ethanol decolorization time, and prevent changes in the structural characteristics of polysaccharides that may be caused by enzyme catalysis.

[0026] 2. The present invention uses microwaves to dry the Longshan lily bulbils at low temperatures and then uses a low-temperature ultrafine grinder to pulverize them. This prevents changes in the structural properties of the polysaccharides caused by high-temperature drying or pulverization. Furthermore, the lily bulbil powder produced by ultrafine grinding has small particle sizes, which facilitates the full dissolution of the polysaccharides.

[0027] 3. The present invention uses a high-temperature resistant amylase for simultaneous treatment in the water extraction step. On the one hand, it can effectively remove starch from the lily bulbils by enzymatic hydrolysis and improve the purity of the polysaccharide; on the other hand, the simultaneous removal of starch can also save extraction time and improve extraction efficiency.

[0028] 4. The present invention first uses water for extraction to extract water-soluble polysaccharides, and then uses an alkaline solution combined with microwaves to extract alkali-soluble polysaccharides to prepare Longshan lily bulbil polysaccharides, and its immune activity is enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the promoting effect of the Longshan lily bulbil polysaccharide prepared in Examples 1-3 and Control Group 1 of the present invention on the secretion of NO by macrophages.

[0030] Figure 2This is the promoting effect of the Longshan lily bulbil polysaccharide prepared in Examples 1-3 and Control Group 1 of the present invention on the secretion of TNF-α by macrophages.

[0031] Figure 3 This is the promoting effect of the Longshan lily bulbil polysaccharide prepared in Examples 1-3 and Control Group 1 of the present invention on the secretion of IL-2 by macrophages. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Example 1

[0033] A method for preparing Longshan lily bulbil polysaccharide comprises the following steps:

[0034] (1) Collect fresh bulbils of Lilium longituba, cut them in half, blanch them in boiling water for 90 seconds to inactivate enzymes, remove them and drain them, dry them in a microwave dryer at 42°C for 45 hours, grind them in a low-temperature ultrafine grinder at 25°C, and pass them through a 300-mesh sieve to obtain Lilium longituba bulbil powder;

[0035] (2) Place the bulbil powder of Lilium truncatum in a bottle, add 5 times the amount (volume-to-mass ratio, the same below) of 95% ethanol solution, decolorize and defatting at 40°C for 6 hours, treat for 3 times in total, filter, evaporate the ethanol from the residue, and dry it at 40°C to prepare the bulbil alcohol-insoluble matter of Lilium truncatum;

[0036] (3) Add 10 times the amount of distilled water and 0.3% of thermostable amylase to the alcohol-insoluble matter obtained in step (2), extract at 75°C for 200 minutes, filter, and set aside the filter residue; after the filtrate is concentrated, extract three times with a mixture of chloroform and n-butanol in a volume ratio of 1:4, collect the upper aqueous solution, add anhydrous ethanol until the volume ratio of ethanol in the solution is 75%, let it stand at 4°C for 8 hours, centrifuge to obtain a precipitate, evaporate the ethanol from the precipitate, and dry it at 40°C to obtain substance A;

[0037] (4) Add 15 times the amount of 3% sodium hydroxide solution to the filter residue obtained in step (3), place it in a microwave digester, extract it 4 times at 400 W power, each time for 20 minutes, cool it to room temperature, filter it, and concentrate the filtrate by rotary evaporation at 45°C. Pour it into a dialysis membrane (molecular weight cutoff 5 kDa), dialyze it in distilled water for 48 hours, and use the dialysate for later use.

[0038] (5) The dialysate obtained in step (4) was extracted three times with a mixture of chloroform and n-butanol in a volume ratio of 1:3, the upper aqueous solution was collected, anhydrous ethanol was added until the volume ratio of ethanol in the solution was 75%, and the solution was allowed to stand at 4°C for 8 hours. The precipitate was obtained by centrifugation and dried at 40°C for 20 hours to obtain lily bulbil polysaccharide. Example 2

[0039] A method for preparing Longshan lily bulbil polysaccharide comprises the following steps:

[0040] (1) Collect fresh bulbils of Lilium truncatum, cut them in half, blanch them in boiling water for 100 seconds to inactivate enzymes, remove them and drain them, dry them in a microwave dryer at 45°C for 40 hours, grind them in a low-temperature ultrafine grinder at 30°C, and pass them through a 400-mesh sieve to obtain Lilium truncatum bulbil powder;

[0041] (2) Place the bulbil powder of Lilium truncatum in a bottle, add 8 times the amount of 95% ethanol solution, decolorize and defatting at 45°C for 4 hours, treat 5 times in total, filter, evaporate the ethanol from the residue, and dry it at 40°C to prepare the bulbil alcohol-insoluble matter of Lilium truncatum;

[0042] (3) Add 15 times the amount of distilled water and 0.8% of thermostable amylase to the alcohol-insoluble matter obtained in step (2), extract at 78°C for 150 minutes, filter, and set aside the residue;

[0043] (4) Add 20 times the amount of 5% sodium hydroxide solution to the filter residue obtained in step (3), place it in a microwave digester, extract it three times at 600 W power, each time for 30 minutes, cool it to room temperature, filter it, and concentrate the filtrate by rotary evaporation at 45°C. Pour it into a dialysis membrane (molecular weight cutoff 5 kDa), dialyze it in distilled water for 48 hours, and use the dialysate for later use.

[0044] (5) The dialysate obtained in step (4) was extracted four times with a mixture of chloroform and n-butanol in a volume ratio of 1:4, the upper aqueous solution was collected, anhydrous ethanol was added until the volume ratio of ethanol in the solution was 78%, and the solution was allowed to stand at 4°C for 10 hours. The precipitate was obtained by centrifugation and dried at 45°C for 18 hours to obtain lily bulbil polysaccharide. Example 3

[0045] A method for preparing Longshan lily bulbil polysaccharide comprises the following steps:

[0046] (1) Collect fresh bulbils of Lilium longituba, cut them in half, blanch them in boiling water for 120 seconds to inactivate enzymes, remove them and drain them, dry them in a microwave dryer at 48°C for 30 hours, grind them in a low-temperature ultrafine grinder at 35°C, and pass them through a 500-mesh sieve to obtain Lilium longituba bulbil powder;

[0047] (2) Place the bulbil powder of Lilium truncatum in a bottle, add 10 times the amount of 95% ethanol solution, decolorize and defatting at 50°C for 2 hours, treat for 6 times in total, filter, evaporate the ethanol from the residue, and dry it at 40°C to prepare the bulbil alcohol-insoluble matter of Lilium truncatum;

[0048] (3) Add 20 times the amount of distilled water and 1.2% of thermostable amylase to the alcohol-insoluble matter obtained in step (2), extract at 85°C for 100 minutes, filter, and set aside the filter residue;

[0049] (4) Add 30 times the amount of 6% sodium hydroxide solution to the filter residue obtained in step (3), place it in a microwave digester, extract it twice at 800 W power, each time for 40 minutes, cool it to room temperature, filter it, and concentrate the filtrate by rotary evaporation at 45°C. Pour it into a dialysis membrane (molecular weight cutoff 5 kDa), dialyze it in distilled water for 48 hours, and use the dialysate for later use.

[0050] (5) The dialysate obtained in step (4) was extracted five times with a mixture of chloroform and n-butanol in a volume ratio of 1:5, the upper aqueous solution was collected, anhydrous ethanol was added until the volume ratio of ethanol in the solution was 80%, the solution was allowed to stand at 4°C for 12 hours, the precipitate was obtained by centrifugation, and the precipitate was dried at 50°C for 15 hours to obtain lily bulbil polysaccharide.

[0051] Control group 1

[0052] On the basis of Example 1, the step (4) of "placing in a microwave digestion instrument and extracting at 400 W power for 4 times, each time for 20 minutes" was changed to "placing in a water bath at 95°C and extracting for 4 times, each time for 20 minutes", and the other steps were consistent with Example 1. The prepared Longshan lily bulbil polysaccharide was used as control group 1.

[0053] Experimental Example 1

[0054] The physicochemical properties of substance A and Longshan lily bulbil polysaccharide were analyzed, including the determination of total polysaccharide content using phenol-sulfuric acid, the analysis of monosaccharide composition and ratio using ion chromatography, and the determination of weight-average molecular weight using gel permeation chromatography-laser light scattering.

[0055] The results of the physicochemical properties of the Longshan lily bulbil polysaccharides in Examples 1 to 3 and Control Group 1 are shown in Table 1.

[0056] Table 1

[0057]

[0058] The above experimental results show that there is no significant difference in the total sugar content of the Longshan lily bulbil polysaccharides prepared in Examples 1 to 3. The total sugar content of the (alkali-soluble) Longshan lily bulbil polysaccharide prepared in Example 1 is significantly higher than that of substance A (Longshan lily bulbil water-soluble polysaccharide). Compared with the control group 1, the total sugar content of the Longshan lily bulbil polysaccharide prepared in Example 1 after microwave treatment is significantly increased.

[0059] The monosaccharide components of the Longshan lily bulbil polysaccharides prepared in Examples 1 to 3 all include xylose, rhamnose, glucose, galactose, and galacturonic acid, and the percentages of the five monosaccharides are not much different. There is a significant difference in the monosaccharide composition of the Longshan lily bulbil polysaccharide prepared by substance A and Example 1, which is manifested in that substance A contains mannose but does not contain xylose, and the proportion of glucose and galacturonic acid in the polysaccharide of Example 1 is lower, while the proportion of galactose in the polysaccharide of Example 1 is higher; compared with the control group 1, the monosaccharide composition of the Longshan lily bulbil polysaccharide prepared in Example 1 after microwave treatment does not change, but the percentages between the components have changed, especially the proportions of rhamnose and glucose decrease, and the proportions of galactose and galacturonic acid increase.

[0060] The weight average molecular weights of the Longshan lily bulbil polysaccharides prepared in Examples 1 to 3 are relatively similar. The molecular weight of substance A is significantly lower than that of Example 1, and the molecular weight of the polysaccharide in Control Group 1 is significantly higher than that of Example 1.

[0061] Experimental Example 2

[0062] With reference to the literature “Xiaona Yu, Xiushi Yang, Bo Cui, Lijun Wang, Guixing Ren. Antioxidant and immunoregulatory activity of alkali-extractable polysaccharides from North American ginseng. International Journal of Biological Macromolecules. 2014, 65: 357-361.”, the immune enhancing activity of the Longshan lily bulbil polysaccharide of the present invention was evaluated by measuring the secretion of macrophage immune factors NO, TNF-α, and IL-2.

[0063] Depend on Figure 1 、 Figure 2 、 Figure 3 It can be seen that the Longshan lily bulbil polysaccharides prepared in Examples 1, 2 and 3 can promote macrophages to secrete NO, TNF-α and IL-2, thereby showing an immune enhancement effect, and the immune enhancement effects of the polysaccharides in the three examples are relatively close.

[0064] The secretion of the three immune factors in Example 1 at each concentration was higher than that of substance A. Compared with control group 1, the secretion of the three immune factors in Example 1 at each concentration was higher than that of control group 1, which indicates that the immunopotency of the polysaccharide extracted by microwave in Example 1 is significantly higher than that in control group 1.

[0065] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A Longshan lily bulbil polysaccharide, characterized by: Its total sugar content is >94%, and the monosaccharide composition is: mannose: rhamnose: glucose: galactose: galacturonic acid = 5.12-5.48: 7.04-7.79: 61.55-62.38: 17.37-18.14: 7.28-7.91, with a weight-average molecular weight of 183916-184735 Da; The preparation method comprises the following steps: S1. Take Longshan lily bulbils, sterilize with boiling water to inactivate enzymes, dry and crush, and pass through a 300-500 mesh sieve to obtain Longshan lily bulbil powder; S2, adding ethanol solution to the Longshan lily bulbil powder, decolorizing and defatting, filtering, and drying the filter residue to obtain Longshan lily bulbil alcohol-insoluble matter; S3, adding water and thermostable amylase to the bulbilol-insoluble matter of Longshan lily, extracting at 75-85° C. for 100-200 minutes, filtering to obtain a residue and a filtrate, and setting the residue aside; S4. Add sodium hydroxide solution to the filter residue obtained in step S3, perform microwave digestion, concentrate the filtrate by rotary evaporation, and dialyze the filtrate for later use; The microwave digestion is specifically as follows: extracting 2 to 4 times at a power of 400 to 800 W, each time for 20 to 40 minutes, cooling to room temperature, and filtering; S5. After the dialyzate is concentrated, it is extracted with a mixture of chloroform and n-butanol, the upper aqueous solution is collected, anhydrous ethanol is added, and the mixture is allowed to stand, followed by centrifugation to obtain a precipitate, which is then dried to obtain Longshan lily bulbil polysaccharide.

2. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that The polysaccharide is extracted from fresh Longshan lily bulbils.

3. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S1, the time for killing the enzyme is 90 to 120 seconds.

4. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S1, the pulverization is performed in a low-temperature ultrafine pulverizer.

5. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S2, the added volume of the ethanol solution is 5 to 10 times the mass of the Longshan lily bulbil powder; and the ethanol solution is an ethanol aqueous solution with a mass fraction of 95%.

6. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S2, the decolorization and degreasing specifically includes: treating at 40-50°C for 2-6 hours, and repeating 3-6 times.

7. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S3, the volume of water added is 10-20 times the mass of the bulbilol-insoluble matter of the longshan lily, and the amount of the thermostable amylase used is 0.2%-1% of the mass of the bulbilol-insoluble matter of the longshan lily.

8. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S4, the added volume of the sodium hydroxide solution is 15-30 times the mass of the filter residue, wherein the concentration of the sodium hydroxide solution is 3wt%~6wt%.

9. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S5, the volume ratio of chloroform to n-butanol in the chloroform and n-butanol mixture is 1:3-5.

10. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S5, anhydrous ethanol is added until the volume proportion of anhydrous ethanol in the solution is 75% to 80%.

11. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S5, the standing temperature is 4° C. and the standing time is 8 to 12 hours.

12. The Longshan lily bulbil polysaccharide according to claim 1, characterized in that In step S5, the drying temperature is 40-50°C.

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